Recombinant human DNase-I improves acute respiratory distress syndrome via neutrophil extracellular trap degradation

Abstract

Background: Respiratory failure is the primary cause of death in patients with COVID-19, whereas coagulopathy is associated with excessive inflammation and multiorgan failure. Neutrophil extracellular traps (NETs) may exacerbate inflammation and provide a scaffold for thrombus formation.

Objectives: The goal of this study was to determine whether degradation of NETs by recombinant human DNase-I (rhDNase), a safe, Food and Drug Administration-approved drug, reduces excessive inflammation, reverses aberrant coagulation, and improves pulmonary perfusion after experimental acute respiratory distress syndrome (ARDS).

Methods: Intranasal poly(I:C), a synthetic double-stranded RNA, was administered to adult mice for 3 consecutive days to simulate a viral infection, and these subjects were randomized to treatment arms, which received either an intravenous placebo or rhDNase. The effects of rhDNase on immune activation, platelet aggregation, and coagulation were assessed in mice and donor human blood.

Results: NETs were observed in bronchoalveolar lavage fluid and within regions of hypoxic lung tissue after experimental ARDS. The administration of rhDNase mitigated peribronchiolar, perivascular, and interstitial inflammation induced by poly(I:C). In parallel, rhDNase degraded NETs, attenuated platelet-NET aggregates, reduced platelet activation, and normalized the clotting time to improve regional perfusion, as observed using gross morphology, histology, and microcomputed tomographic imaging in mice. Similarly, rhDNase reduced NETs and attenuated platelet activation in human blood.

Conclusion: NETs exacerbate inflammation and promote aberrant coagulation by providing a scaffold for aggregated platelets after experimental ARDS. Intravenous administration of rhDNase degrades NETs and attenuates coagulopathy in ARDS, providing a promising translational approach to improve pulmonary structure and function after ARDS.

Keywords: coagulation; immunothrombosis; inflammation; platelets; thrombosis.

Figure 1

Increased neutrophil infiltration and neutrophil extracellular trap (NET) formation in the lung and bronchoalveolar lavage fluid (BALF) after experimental acute respiratory distress syndrome. (A) Representative flow cytometry plots showing activation of CD45⁺ Ly6G⁺ neutrophils in the blood, lung tissue, or BALF on experimental day 5. Poly(I:C) mice were treated with either saline (placebo) or 5-mg/kg recombinant human DNase-I (rhDNase) (intravenous). Data are representative of 5 to 7 mice per group. (B) Quantification of poly(I:C)-induced neutrophil activation and NET formation in the lung, BALF, and blood after administration of placebo or rhDNase. Scatterplots represent 5 to 7 mice per group. Data were compared using 1-way analysis of variance, followed by the Tukey post hoc test. ∗p < .05, ∗∗p < .01, ∗∗∗∗p < .0001. (C) Immunohistochemistry of Ly6G⁺ Cit-H3⁺ NETs in lung tissue after administration of placebo or rhDNase. 4′,6-diamidino-2-phenylindole was used as a counterstain. Scale bar = 100 μm. Mean fluorescence intensity from 25 random fields from 5 mice per group was quantified and presented as mean ± SD. Data were compared using 1-way analysis of variance, followed by the Tukey post hoc test. ∗p < .05, ∗∗p < .01, ∗∗∗∗p < .0001. DAPI, 4′,6-diamidino-2-phenylindole; FSC, forward scatter; MFI, mean fluorescence intensity; SSC, side scatter.

Figure 2

Recombinant human DNase-I (rhDNase) reduces neutrophil infiltration and improves lung morphology after experimental acute respiratory distress syndrome. (A) Gross images of lung tissue on experimental day 6. Poly(I:C) mice were treated with intravenous administration of either saline (placebo) or 5-mg/kg rhDNase. Bilateral regions of hyperemia are denoted by black dotted lines. Regions of pale tissue, indicative of hypoperfusion and regional hypoxia, are labeled with blue dotted lines. Lung injury was observed across the lung lobes from placebo-treated mice, with less pronounced effects observed after rhDNase treatment. Scale bar = 5 mm. (B) Histologic assessment of lung tissues. Data are representative of 6 mice per group. Top panel: Note that the increased cellularity observed in the poly(I:C) group, consistent with inflammatory activation, was attenuated following rhDNase treatment. Scale bar = 200 μm. Middle panel (second and third rows): Peribronchiolar and perivascular inflammation was observed in the poly(I:C) group. These changes were reduced with rhDNase treatment. Scale bar = 50 μm. Bottom panel: Interstitial inflammation and diffuse alveolar damage, a feature associated with early stages of acute respiratory distress syndrome, were seen in the poly(I:C) group. rhDNase treatment mitigated interstitial inflammation. Scale bar = 50 μm. (C) Confocal microscope images of poly(I:C)-treated lung tissue on experimental day 6. Ly6G⁺ infiltrating neutrophils localized in regions of tissue hypoxia, as indicated by increased hypoxyprobe-1 fluorescence. Scale bar = 100 μm. The mean fluorescence intensity was quantified from 25 random fields from 5 mice per group. Scatterplots were compared using 1-way analysis of variance, followed by the Tukey post hoc test. ∗∗∗p < .001, ∗∗∗∗p < .0001. DAPI, 4′,6-diamidino-2-phenylindole; MFI, mean fluorescence intensity.

Figure 3

Recombinant human DNase-I (rhDNase) improves pulmonary perfusion after experimental acute respiratory distress syndrome. (A) Ex vivo microcomputed tomography imaging of the lungs on experimental day 6. Poly(I:C) mice were treated with intravenous administration of either sterile phosphate-buffered saline or 5-mg/kg rhDNase. Representative images depict volume-rendering imaging and maximal intensity project. For volume-rendering imaging, every voxel is assigned an emission color and opacity, which indicates the intensity of the contrast dye within the vasculature. All lungs were scanned using identical parameters, and the same log-scaled histogram was used to color map the lung vasculature. Red color represents lower-intensity structures (vessels with lower contrast in their lumen), blue color represents higher-intensity structures (vessels with higher contrast in their lumen), and green is the range in between those 2. Dorsal, ventral, side, and transverse views are depicted and show perfusion deficits in the poly(I:C)-treated mice. Following rhDNase treatment, perfusion of smaller-caliber blood vessels was observed. (B) Quantification of vascular volume, capillary network, number of blood vessels (BVs), BV lumen thickness, and BV separation, as assessed using microcomputed tomography. rhDNase improved vascular volume, capillary network, and the number of BVs to sham-injured levels. Quantified data are from 6 mice per group and were analyzed using 1-way analysis of variance, followed by the Tukey post hoc test. ∗p < .05, ∗∗p < .01. MIP, maximum-intensity projection; VRI, volume-rendering imaging.

Figure 4

Recombinant human DNase-I reduces platelet activation in blood and lung tissue after experimental acute respiratory syndrome. (A) Quantification of platelet activation in blood, lung tissue, or bronchoalveolar lavage fluid on experimental day 5. Poly(I:C) mice were treated with intravenous administration of either sterile phosphate-buffered saline or 5-mg/kg recombinant human DNase-I. Top panels depict flow cytometry plots to differentiate CD41⁺ (resting) and CD62P⁺ (activated) platelets. (B) Quantification of platelet activation data from 7 mice per group was analyzed using 1-way analysis of variance, followed by the Tukey post hoc test. ∗p < .05, ∗∗p < .01, ∗∗∗p < .001, ∗∗∗∗p < .0001. BALF, bronchoalveolar lavage fluid; FSC, forward scatter.

Figure 5

Recombinant human DNase-I attenuates activated platelet-neutrophil extracellular trap (NET) macroaggregates in lung tissue after experimental acute respiratory distress syndrome. Association of activated platelets (CD41⁺ CD62p⁺) with NETs in lung tissue on experimental day 5, as assessed using flow cytometry. Poly(I:C) mice were treated with intravenous administration of either sterile phosphate-buffered saline or 5-mg/kg recombinant human DNase-I. Quantification of platelet-NET aggregates from 7 mice per group was analyzed using 1-way analysis of variance, followed by the Tukey post hoc test. ∗∗∗∗p < .0001. FSC, forward scatter; SSC, side scatter.

Figure 6

Recombinant human DNase-I (rhDNase) normalizes the clotting time after experimental acute respiratory distress syndrome. (A) Ex vivo quantification of the clotting time of whole blood collected from mixed-sex mice on experimental day 5. Administration of poly(I:C) reduced the clotting time in placebo-treated mice, whereas rhDNase treatment normalized the clotting time to control levels. Data were compared using 1-way analysis of variance (ANOVA), followed by the Tukey post hoc test. ∗∗p < .01. (B) Sex-dependent changes in clotting time were observed following experimental acute respiratory distress syndrome, with males exhibiting a more robust response to poly(I:C)-induced clotting compared with female mice. Data were compared using 1-way ANOVA, followed by the Tukey post hoc test. ∗∗p < .01, ∗∗∗p < .001. Quantification of (C) neutrophil extracellular traps and (D) platelet activation in healthy donor human blood following poly(I:C) treatment ex vivo. Poly(I:C) increased the number of activated platelets, with a concomitant reduction in resting platelets. Treatment with rhDNase normalized platelet activity to untreated control levels. Data were analyzed using 1-way ANOVA, followed by the Tukey post hoc test. ∗∗p < .01, ∗∗∗p < .001, ∗∗∗∗p < .0001. FSC, forward scatter; NET, neutrophil extracellular trap; SSC, side scatter.

Supplemental Figure 1

rhDNase suppresses inflammatory activation after experimental ARDS. (A) Quantification of lymphocyte activation in lung tissue, BALF, or blood at experimental d5. Poly(I:C) mice were treated with intravenous injections of either sterile PBS or 5 mg/kg rhDNase. Data were analyzed using flow cytometry and are representative of n=3-6 mice/group. (B) Quantification of poly(I:C)-induced inflammatory cytokine (IL-6, IL-17, IL-10) expression in lung, BALF, and blood after administration of placebo or rhDNase. Data were analyzed using flow cytometry and scatterplots represent n=6-8 mice/group. For all panels, data were compared by one-way ANOVA followed by Tukey’s post hoc test. ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001, ∗∗∗∗p<0.0001.

Supplemental Figure 2

Plasma DNase-I levels are increased at day 5 following rhDNase intravenous administration. (A) Plasma was collected from randomly selected mice from each group at experimental day 5. Western blots were probed for DNase-I. (B) Total protein loading was visualized by Ponceau S staining. (C) DNase-I expression was normalized to total protein. Densitometry analysis revealed the increased expression of plasma DNase-I in poly(I:C) mice treated with intravenous 5mg/kg rhDNase, as compared to sham and poly(I:C) mice treated with saline (placebo). Data are representative of n=3 mice/group and were compared by one-way ANOVA followed by Tukey’s post hoc test. ∗∗p<0.01.